We recently reported that CTLA-4, a molecule previously thought to be expressed only by T cells, is also expressed by pituitary endocrine cells. This finding has opened a new area of investigation in pituitary autoimmunity, and it is clinically relevant because therapeutic injections of CTLA-4 blocking antibodies, used as a form of cancer immunotherapy, cause secondary hypophysitis in a significant percentage of the patients (about 12%). There is a fundamental gap in the understanding of the role of pituitary CTLA-4, and in the ability to diagnose and treat hypophysitis. The long-term goals of our research program are to elucidate the pathophysiological role of pituitary CTLA-4 and to prevent secondary hypophysitis. The goals of this application are to identify factors that modulate the expression of pituitary CTLA-4 and to develop a non-invasive and reliable serum test based on pituitary antibodies for diagnosis of hypophysitis. In fact, although hypophysitis was first reported in 1962 in its primary form and in 2003 in the form secondary to cancer immunotherapy, such a diagnostic test is not currently available. Our central hypothesis is that CTLA-4 functions in the pituitary gland as an immunological shield. Under normal conditions, the shield prevents infiltration of autoreactive lymphocytes, thus establishing the pituitary gland as an immuno-privileged site and explaining the rarity of primary hypophysitis. In the context of cancer immunotherapy, however, the same pituitary CTLA-4 shield becomes the target of the injected therapeutic anti-CTLA-4 antibody, thus initiating an autoimmune reaction that damages the pituitary cells. The rationale for the proposed research is that understanding the function of pituitary CTLA-4 and the associated autoimmune phenomena will lead to innovative approaches for the diagnosis and treatment of hypophysitis. Guided by strong preliminary data, we will test our hypothesis using two specific aims. Aim 1 focuses on elucidating the role and regulation of CTLA-4 expression in pituitary endocrine cells, using banked and prospective human pituitary tissues (1A), pituitary cell lines in vitro (1B), and functional studies in mice (1C). Aim 2 focuse on improving the diagnosis and treatment of hypophysitis secondary to CTLA-4 blockade, by discovering pituitary autoantigens using Phage ImmunoPrecipitation sequencing technology (2A), by establishing the analytical (2B) and clinical (2C) validity of antibody-based diagnostic tests, and by identifying small bioactive molecules potentially capable of modulating pituitary CTLA- 4 expression (2D). Our research is innovative because it opens a new area of investigation centered on pituitary CTLA-4, and uses a high throughput autoantigen identification methodology as the foundation for developing novel diagnostic tools. The research is significant because it will enhance human health by improving diagnosis of hypophysitis and generating fundamental knowledge about pituitary CTLA-4.